Abstract We describe the isolation and initial character- ization of KlCOX18, a gene that is essential for the as- sembly of a functional cytochrome oxidase in the yeast Kluyveromyces lactis. Cells carrying a recessive nuclear mutation in this gene are respiratory deficient and contain reduced levels of cytochromes a and a 3 . The KlCOX18 gene has been cloned by complementation of the respec- tive nuclear mutation, sequenced, and disrupted. KlCOX18 is located on chromosome II and contains an open reading frame of 939 base pairs. The corresponding protein exhibits 70.4% similarity to the Cox18p of Sac- charomyces cerevisiae. It contains three possible mem- brane-spanning domains and a putative amino-terminal mitochondrial import sequence. The strain carrying a null mutation in KlCOX18 does not grow on non-fermentable carbon sources and is deficient in both cytochrome c ox- idase and respiratory activity. It is proposed that KlCox18p, like its S. cerevisiae counterpart, provides an important function at a later step of the cytochrome oxi- dase assembly pathway. Key words Kluyveromyces lactis · Cytochrome c oxidase · Mitochondria · Yeast Introduction Cytochrome c oxidase is the terminal enzyme complex of the mitochondrial and bacterial respiratory chains. This membrane protein catalyzes the final step of aerobic res- piration transferring electrons from ferrocytochrome c to molecular oxygen, with the simultaneous formation of a proton gradient. Active preparations of yeast cytochrome c oxidase contain a total of nine subunits: three encoded by mitochondrial genes (COX1, COX2 and COX3, respec- tively) and six encoded by nuclear genes (COX4, COX5a or COX5b, COX6, COX7, COX9 and COX8, respectively) (Poyton et al. 1995). As the enzyme complex is formed by the contribution of two genetic systems of eucaryotic cells, cytochrome c oxidase from Saccharomyces cerevisiae has been a useful model system for studying both the cross talk between nuclear and mitochondrial genomes and subunit structure-function relationship in eucaryotic cytochrome c oxidases. The studies on respiration-deficient yeast mu- tants have shown that, in addition to the COX structural genes, the biogenesis of cytochrome c oxidase depends also on a large number of other nuclear genes (McEwen et al. 1986; Nobrega et al. 1990; Tzagoloff and Dieckmann 1990; Tzagoloff et al. 1990). The function of all of these genes has not yet been elucidated. Some of them have been shown to be required for the expression of the mitochondrial COX genes, others for heme A synthesis and/or the assembly of a functional holoenzyme (Schultze and Rodel 1989; McEwen et al. 1993; Bonnefoy et al. 1994; Church et al. 1996). The gene sequence data from Kluyveromyces lactis mit- ochondrial, as well as nuclear DNA are still fragmentary. The genes for cytochrome oxidase subunit 1 (Hardy and Clark-Walker 1991) and subunit 2 (Hardy and Clark- Walker 1990) have already been sequenced. Here, we report the isolation and characterization of the KlCOX18 gene whose encoded protein, homologous to Cox18p in S. cerevisiae, appears to be required for cyto- chrome oxidase biogenesis. Curr Genet (1997) 32: 267–272 © Springer-Verlag 1997 Received: 25 March / 3 July 1997 Imrich Hikkel · Yvetta Gbelská Quirina J. M. van der Aart · Gert Lubec Július S ˇ ubík Cloning and characterization of KlCOX18, a gene required for activity of cytochrome oxidase in Kluyveromyces lactis ORIGINAL PAPER I. Hikkel · Y. Gbelská · J. S ˇ ubík () Department of Microbiology and Virology, Comenius University, Mlynská dolina B2, 842 15 Bratislava, Slovak Republic Fax: +4217729064 e-mail: subik@fns.uniba.sk Q. J. M. van der Aart Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 Al Leiden, The Netherlands G. Lubec Department of Paediatrics, University of Vienna, 1090 Vienna, Austria Communicated by L. Frontali